tips and tricks hplc troubleshooting
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hplcTRANSCRIPT
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Tips and Tricks of HPLC System Troubleshooting
Agilent Technologies, Inc.LC Tips And Tricks Seminar Series
Page 2
Trouble Shooting Steps
You Have Recognized There is a Problem! How Do You Fix It?
•1st Did System Suitability or Sample Fail? •2nd Review Method for Compliance
– Is The Procedure Being Followed Properly?– Are Instrument Settings Correct?
•3rd Ask More Questions!– When Did the System Last Function Properly?– Has Anything Been Changed?
•4th Review ALL parameters!– The Obvious Is Not Always the Cause– Was There More Than One Change?
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PumpInjector/AutosamplerColumnDetectorData System/Integrator
HPLC System Components
Problems Can Be Related to All Components in the System
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Categories of Column and System Problems
A. Pressure
B. Peak shape
C. Retention
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Column Observations Potential Problems
High pressure - Plugged frit
- Column contamination
- Plugged packing
Low Pressure - Leak
- Flow Incorrect
Pressure Issues
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Determining the Cause and Correcting High Back Pressure
• Check pressure with/without column - many pressure problems are due to blockages in the system or guard col.
•Remove Column - Pressure Still High?
•Remove Guard – Pressure Still High?
•If Column pressure is high:
• Back flush column – Clear “dirty” frit surface • Wash column – Eliminate column contamination
and plugged packing– high molecular weight/adsorbed
compounds– precipitate from sample or buffer
Change frit – Clear plugged frit PREVENT THIS!
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Column Cleaning
Use at least 25 mL of each solvent for analytical columns
Flush with stronger solvents than your mobile phase.
Reversed-Phase Solvent Choices in Order of Increasing Strength
• Mobile phase without buffer salts• 100% Methanol• 100% Acetonitrile• 75% Acetonitrile:25% Isopropanol• 100% Isopropanol• 100% Methylene Chloride* • 100% Hexane*
*Tip: When using either Hexane or Methylene Chloride the column must be flushed with Isopropanol before returning to your reversed-phase mobile phase.
Must Reverse to
Re-Equilibrate
This Is Time Consuming
Often Performed Offline
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Changing a Frit May Not Be a Good IdeaMay not be possible with new generation columns
May damage high performance columns
ColumnInlet Frit
CompressionFerrule
Column Body
Female End Fitting
Male End Fitting
Wear glovesDo not allow bed to dryDo not touch the column - body heat will extrude packingDo not overtighten
Tip: Prevention is a Much Better Idea!
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The Trick: Prevention Techniques - A Better Choice!
• Use column protection- In-line filters - Guard columns
• Filter samples • Filter buffered mobile phases
• Sample clean-up (i.e. SPE) • Appropriate column flushing
Easy
Not As Easy
Page 10
Inexpensive Filters Prevent Column Frit Plugging Regenerated Cellulose (RC) Recommended•Universal hydrophilic membrane, compatible with most solvents - aqueous and organic•High purity, extremely low extractables and binding•More Uniform Surface•Different than Other Cellulose Filters!!
In-line Filters Easy to Use and replace
Frits Available in 0.2,0.5 and 2.0µ Porosity
Much Less expensive than a Column
Easier and Faster to Replace than a Column Frit
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What Are Common Peak Shape Issues?
1. Split peaks
2. Peak tailing
3. Broad peaks
• Many peak shape issues are also combinations - i.e. broad and tailing or tailing with increased retention
•Symptoms do not necessarily affect all peaks in the chromatogram
•Each of these problems can have multiple causes
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Peak Splitting Caused By Disrupted Sample Path
Split or Double Peaks
Normal Double Peaks
Tip: Similar Effect Can be Caused by Partially Plugged Frit
•Flow Path Disrupted by Void
•Sample Allowed to Follow Different Paths Through Column
•Poorly Packed Bed Settles in Use
•High pH Dissolves Silica
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0 5 10 15
1
3
4
2
Time (min)0 5 10 15
1
3
4
2
Time (min)0 5 10 15
1
3
4
2
Time (min)
Split Peaks from Column Contamination
Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH Flow Rate: 1.0 mL/min Temperature: 35°C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine
Injection 1 Injection 30 Injection 1After Column Washwith 100% ACN
Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the columnHow could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash)
Group/Presentation TitleAgilent Restricted
September 10, 2008Month ##, 200X
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Split Peaks from Injection Solvent Effects
Column: StableBond SB-C8, 4.6 x 150 mm, 5 μm Mobile Phase: 82% H2O : 18% ACNInjection Volume: 30 μL Sample: 1. Caffeine 2. Salicylamide
A. Injection Solvent100% Acetonitrile
B. Injection SolventMobile Phase
Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape problems such as peak splitting or broadening
Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase
0 10Time (min)
1
2
0 10Time (min)
1
2
8
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Peak Tailing, Broadeningand Loss of Efficiency
May be caused by:
• Column “secondary interactions”
• Column contamination• Column aging• Column loading• Extra-column effects
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Normal Tailing
Normal Tailing
Symmetry > 1.2
All Peaks Tail:
Extra-Column Effects.Build up of Contamination on Column Inlet.Heavy Metals.Bad Column.
CausesSome Peaks Tail:
Secondary - Retention Effects.Residual Silanol Interactions.Small Peak Eluting on Tail of Larger Peak.
Peak Shape: Tailing Peaks
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Peak TailingIdentifying Column “Secondary Interactions”
Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange sites at mid-range pH values
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 pH 7.0 : 15% ACN Flow Rate: 1.0 mL/minTemperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
No TEAUSP TF (5%)
1. 1.292. 1.913. 1.634. 2.355. 1.57
10 mM TEAUSP TF (5%)
1. 1.192. 1.183. 1.204. 1.265. 1.14
TIme (min) Time (min)0.0 2.5 5.0
54
32
1
54
32
1
0.0 2.5 5.0
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Peak Tailing
Low pH Minimizes “Secondary Interactions”for Amines
Column: Alkyl-C8, 4.6 x 150 mm, 5μm Mobile Phase: 85% 25 mM Na2HPO4 : 15% ACN Flow Rate: 1.0 mL/minTemperature: 35°C Sample: 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine
pH 3.0USP TF (5%)
4. 1.33
pH 7.0USP TF (5%)
4. 2.35
Tip: Reducing mobile phase pH reduces interactions with silanols and peak tailing.
Time (min)0.0 2.5 5.0
54
32
1
54
32
1
Time (min)0.0 2.5 5.0
10
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0 5
1
2,3
4
5
Time (min)
7
6
0 5 10
1
2
3
4
Time (min)
6
5
7
Peak Tailing
High pH Eliminates “Secondary Interactions” for Amines
Peak Shape and Retention of this sample of basic compounds improves at high pH where column has high IEX activity. Why?
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m Mobile Phase: See Below Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm
Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. DiphenhydraminepH 730% 20 mM Na2HPO470% MeOH
pH 1130% 20 mM TEA70% MeOH
tR = 8.5 tR = 11.4
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0.0 2.5 5.0
2
41
3
Time (min)
2
41
3
0.0 2.5 5.0Time (min)
2
41
3
0.0 2.5 5.0Time (min)
Peak Tailing - Column Contamination
Column: StableBond SB-C8, 4.6 x 250 mm, 5μm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/minTemperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene
Plates TF
1. 7629 2.082. 12043 1.643. 13727 1.694 13355 1.32
Plates TF
1. 7906 1.432. 12443 1.213. 17999 1.194 17098 1.25
Plates TF
1. 7448 1.062. 12237 1.213. 15366 1.114 19067 1.17
QC test forward direction
QC test reverse directionQC test after cleaning
100% IPA, 35°C
Tip: Quick Test to Determine if Column is Dirty or Damaged
Trick: Reverse Column and Run Sample –If Improved, Possible Cleaning Will Help -No improvement-Column Damaged and Needs to be Replaced
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Causes:
Column Overload
Normal FrontingSymmetry < 0.9
2000
1500
1000
500
0
0 5 10 15 20 25
Time (min)
mA
U
Peak Shape: Fronting Peaks
Group/Presentation TitleAgilent Restricted
September 10, 2008Month ##, 200X
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Peak Tailing/Broadening Sample Load Effects
Columns: 4.6 x 150 mm, 5μm Mobile Phase: 40% 25 mM Na2HPO4 pH 7.0 : 60% ACN Flow Rate: 1.5 mL/minTemperature: 40°C Sample: 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine
Broadening Competitive C8
Plates
A.
B.
C.
D.
High Loadx10
Low Load
C D1. 850 59412. 815 78423. 2776 62314. 2539 83595. 2735 100226. 5189 10725
Tailing Eclpse XDB-C8USP TF (5%) i
A B1. 1.60 1.702. 2.00 1.903. 1.56 1.564. 2.13 1.705. 2.15 1.866. 1.25 1.25
0 5 10Time (min)
0 5 10Time (min)
0 5Time (min)
0 5Time (min)
Tip: Evaluate Both Volume and Mass Loading
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Peak Shape: Broad Peaks
All Peaks Broadened:
• Loss of Column Efficiency.• Column Void.• Large Injection Volume.
Some Peaks Broadened:
• Late Elution from Previous Sample (Ghost Peak).– High Molecular Weight.– Sample - Protein or Polymer.
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Time (min)
3
1
2
0 5 10 15
Unknown “Phantom” Peaks
Column: Extend-C18, 4.6 x 150 mm, 5 μm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/minTemperature: R.T. Detection: UV 254 Sample: 1. Maleate 2. Pseudoephedrine 3. Chlorpheniramine
Plates1. 59222. 98793. 779
Tip: The extremely low plates for moderately retained peaks are an indication of a very late eluting peak from a preceding run.
Time (min)
1
0 5 10
Sample 1: Chlorpheniramine maleatePeak 1: maleate
Sample 2 : Chlorpheniramine maleate
and PseudoephedrinePeak 1: maleatePeak 2: pseudoephedrinePeak 3: chlorpheniramine (from 1st
injection)
“Phantom” peak fromfirst injection
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Use short, small internal diameter tubing between the injector and the column and between the column and the detector.
Make certain all tubing connections are made with matched fittings.
Use a low-volume detector cell.
Inject small sample volumes.
Increasing Extra-Column Volume
Extra-Column Dispersion
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Peak Broadening Extra-Column Volume
Column: StableBond SB-C18, 4.6 x 30 mm, 3.5 μm Mobile Phase: 85% H2O with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame
10 mL extra-columnvolume
50 mL extra-columnvolume (tubing)
Time (min)0.0 0.5 1.0 1.5 2.0
4
32
1
Time (min)0.0 0.5 1.0 1.5 2.0
4
32
1
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Tip: Poorly Made HPLC System Connections Can Cause Peak Broadening
The System Has Been Optimized and :– All Tubing Lengths Are Minimum– Smallest Diameter Tubing Used– Proper Flow Cell Volume
Symptom Still Seems to Have Too Much Extra-Column
Volume
What Is Wrong?
Have You Made the Connections Properly?
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Column Connectors Used in HPLC
0.090 in.
0.130 in.
0.090 in.
0.170 in.
Swagelok Waters
Parker Rheodyne
Valco Uptight0.090 in.
0.080 in.
Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988)
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What Happens If the Connections Poorly Made ?
If Dimension X is too long, leaks will occur
Ferrule cannot seat properly
Mixing Chamber
If Dimension X is too short, a dead-volume, or mixing chamber, will occur
Wrong … too long
Wrong … too shortX
X
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Stainless Steel and Polymer FittingsWhich type is used and when?Stainless Steel (SS) fittings are the best choice for
reliable high pressure sealing• Agilent uses Swagelok type fittings with front and back
ferrules – which give best sealing performance –throughout all our LC systems
PEEK (<400b bar System Pressure) fittings are ideal where:• Connections are changed frequently, i.e. connecting
columns• Pressure is less critical
PolyKetone • Easy, hand tighten column connection • 600 bar Pressure Rating PN: 5042-8957 (10/pk)• Fits to SS Tubing
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Changes in Retention Can Be Chemical or Physical
May be caused by:
• Column aging• Column contamination• Insufficient equilibration• Poor column/mobile phase combination• Change in mobile phase• Change in flow rate• Different Gradient Delay Volumes
Group/Presentation TitleAgilent Restricted
September 10, 2008Month ##, 200X
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Column Aging/Equilibration CausesRetention/Selectivity Changes
Column 1 - After Cleaning with 1% H3PO4 /Equilibration
• The primary analyte was sensitive to mobile phase aging/ conditioning of the column
• The peak shape was a secondary issue (metal chelating compound) resolved by “de-activating” the active metal contamination
Column 1 - Next DayColumn 1 - Initial
0 3 5 9 12 15Time (min)
2
1
0 3 5 9 12 15Time (min)
2
1
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Metal Sensitive Compounds Can Chelate
C O
OH
HM +2
OH
::
M +2C O
OHC N
N: M +2 ::
:
O
:
OH + M +2
CH ::
:
Hint: Look for Lone Pair of Electrons on :O: or N Which Can Form 5 or 6 MemberedRing with Metal
:
Salicylaldehyde 6-membered ring complex
8-hydroxyquinoline5-membered ring complex
a-benzoinoxomine5-membered ring complex
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Acid Wash Can Improve Peak Shape
OH
OH
OHHOOHHO OH
OH1. 2. 1. 2.
Columns: ZORBAX SB-Phenyl4.6 x 150 mm
Mobile Phase: 75% 25 mM ammonium phosphate buffer
25% ACNFlow Rate: 1.0 mL/min.Temperature: RTSample Size: 5 mL
1
1
2
2
Tf: 1.2Tf: 3.7
Before Acid Wash After Acid Wash50 – 100 mLs 1% H3PO4
• A 1% H3PO4 solution is used on SB columns, 0.5 % can be used on endcapped columns.
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Group/Presentation TitleAgilent Restricted
September 10, 2008Month ##, 200X
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Example: Change in Retention/SelectivityUnintended Mobile Phase Variation
Tip: The Source of the Problem is Often Not the Obvious Change
“I have experimented with our mobile phase, opening new bottles of all mobile phase components. When I use all fresh ingredients, the problem ceases to exist, and I have narrowed the problem to either a bad bottle of TEA or phosphoric acid. Our
problem has been solved.”
Column 1 Column 2 - Fresh mobile phase
Column 2
0 4 6Time (min)
0 2 3 4 5 6 7Time (min)
0 4 6Time (min)
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0 10 20 30 40
Tip: Dwell Volume Differences Between Instruments Can Cause Changes in Retention and Resolution
0 10 20 30 40
VD = 0.43 mL Column: ZORBAX Rapid Resolution Eclipse XDB-C84.6 x 75 mm, 3.5 µm
Mobile Phase: Gradient, 0 - 100 %B in 52.5 min.A: 5/95 methanol/ 25 mM phosphatepH 2.50
B: 80/20 methanol/25 mM phosphatepH 2.50
Flow Rate: 0.5 mL/minTemperature: 25°CInjection: 5 μLDetection: 250 nmSample: Mixture of antibiotics and
antidepressantsUpper trace simulates actual run data entered into DryLab® 3.0 softwareLower trace is simulated chromatogram for larger VD
VD = 2.0 mL
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If VD1 > VD2Compensate for longer VD1 by adding
an isocratic hold to VD2, such thatHold + VD2 = VD1
If VD1 < VD2Delay injection, such that VD2 - delay = VD1
001014P1.PPT
Trick: Measure and Correct for Dwell Volume (VD)
Page 38
Mobile Phase pH and pH BuffersWhy Are These So Important in HPLC?•pH Effects Ionization
– Silica Surface of Column– Sample Components of Interest
• Buffers– Resist Changes in pH and Maintain Retention– Improve Peak Shape for Ionizable Compounds
• Effects Column Life– Low pH strips Bonded Phase– High pH Dissolves Silica
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Minimize Change in Retention/SelectivityLot-to-Lot
• All causes of column-to-column change*• Method ruggedness (buffers/ionic strength)• pH sensitivity (sample/column interactions)
*All causes of column-to-column change should be considered first,especially when only one column from a lot has been tested.
Evaluate:
Group/Presentation TitleAgilent Restricted
September 10, 2008Month ##, 200X
Page 40
0 2 4 6 8 10 12 14 16 18Time (min)
2-Base3
4-Base
1
0 2 4 6 8 10 12 14 16 18Time (min)
2
3
4
1
Lot-to-Lot Selectivity Change Related to pH Choice
• pH 4.5 shows selectivity change from lot-to-lot for basic compounds• pH 3.0 shows no selectivity change from lot-to-lot• Indication of poorly controlled ionization
pH 4.5 - Lot 1 pH 3.0 - Lot 1
pH 4.5 - Lot 2 pH 3.0 - Lot 2
0 2 4 6 8 10 12 14 16 18Time (min)
2-Base
3
4-Base
1
0 2 4 6 8 10 12 14 16 18Time (min)
2
3
4
1
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Why Worry About pH?pH, pKa and Weak Acids
At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratioof 1:1. Peak shape can be poor
At pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.
RCOOH RCOO- + H+
Ka = 6.4 x 10-5
pKa = 4.2
Ka =[RCOO-][H+]
[RCOOH]
+ H+
COOH COO_
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Effect of pH on Peak Shape at or Near the Sample pKa
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10Time (min) Time (min)
Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm Mobile Phase: 40% 5 mM KH2PO4: 60% ACN Flow Rate: 1.0 mL/min. Temperature: RT
pH 4.4 pH 3.0
IbuprofenpKa = 4.4
CH3CHCOOH
CH2CH(CH3)2
• Inconsistent and tailing peaks may occur when operating close to an analytepKa and should be avoided.
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Why Worry About pH?pH, pKa and Weak Bases
At pH 9 – the sample exists as protonated and unprotonated diphenhydramine in a ratio of 1:1. Peak shape can be poor.
At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.At pH 8 – 91% of the sample exists as protonated diphenhydramine.
Ka =[R3N][H+][R3NH+]
Ka = 1 x 10-9
pKa = 9
R3NH+ R3N + H+
CHOCH CH N2 2
CH3
CH3
∅
∅
+H
CH3
CH3
∅
∅
CHOCH CH N2 2 + H+
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pH vs. Selectivity for Acids and Bases
5
SCD
++
2 +
1
1.5
1.0
0.5
0.0
-0.5
log k«
3 4 5 6 7 8 pH
A B C
2
4
5
3
3 5 7 9ELUENT pH
40
30
20
10
10
Reten
tion
+++
3
1 7,12 - OC
UDCSOC4
6
C12-OC
J.C. 268(1983) 1
J.C. 111(1975) 149
Column: Nucleosil-C18Mobile Phase: 45% ACN/55% phosphate bufferSample: Bile Acids
Column: mBondapak-C18Mobile Phase: 60% 25 mM phosphate buffer
40% Methanol1. Salicylic acid2. Phenobarbital3. Phenacetin4. Nicotine5. Methamphetamine
• Retention and selectivity can change dramatically when pH is changed.
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Importance of pH and Buffers A Practical Example
•Why the Sample Dictates Use
•What Happens When Buffer Used Effectively
•What Happens When Buffer Ignored or Used Improperly
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Importance of pH and Buffers - A Practical Example Optimized Isocratic Conditions for Cardiac Drugs
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Time (min)
5
4
32
1
Column: StableBond SB-C18, 4.6 x 150 mm, 5 mmMobile Phase: 45% 25 mM NaH2PO4, pH 3.0
55% MeOHFlow Rate: 2.0 mL/min.Temperature:35°CDetection: UV 254 nmSample: Cardiac Drugs
1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine
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I Don’t Have Time to Make Buffers or Adjust pH …
0 5 10 15 20 25Time (min)
Column: StableBond SB-C184.6 x 150 mm, 5 mm
Mobile Phase: A: 20% H2OB: 80% MeOH
Flow Rate: 1.0 mL/min.Temperature:35°CUV Detection: 254 nmSample: Cardiac Drugs
• Buffers are critical to good retention and peak shape in many separations.
Even at very high % MeOH Most Components Strongly Retained with Poor peak Shape Due to IEX at Surface
Page 48
What If You Work Outside the Buffer Range?
0 5 10 15 20 25
5
1
2
34
Time (min)
Columns: StableBond SB-C184.6 x 150 mm, 5 mm
Mobile Phase: A: 30% 25 mM NaH2PO4, pH 4.8 unbuffered
B: 70% MeOHFlow Rate: 1.0 mL/min.Temperature:35°CUV Detection: 254 nmSample: Cardiac Drugs
1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine
Unsuitable Peak Shape
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Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column Lifetimelow pH and high temperature (pH 0.8, 90°C)
000023P1.PPT
Purge Solvent: 50% methanol/water with
1.0% TFASolute: Toluene
Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, 32 (1994) 473-480.
Page 50
Don’t Forget - Match Column to pH of Mobile Phase for Maximum Column LifetimeHigh pH and Room Temperature (pH 11 RT)
Tip: Use Columns Designed for chosen pH
Mobile Phase: 50%ACN: 50% Water : 0.2% TEA(~ pH 11)
After 30 injections
Initial
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Detection Issues
Recognize Where the Problem Originates• Is it a consequence of technique?• Is It expected due to use of certain mobile phase
components?• Can it be corrected by adjusting detector
parameters?• Answers Will Help Find a Solution!Let’s Explore Some Problems and Solutions
Page 52
Causes:
Absorbance of sample is less than the mobile phase.Equilibrium disturbance when sample solvent passes through the column.Normal with Refractive Index Detectors.
Normal Negative
Peak Shape: Negative Peaks
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Ghost Peaks
20% - 100% MeOH GradientNo Sample Injected
Ghost Peaks - Peaks which appear even when no sample is injected.
Problem - Dirty Mobile Phase
60
15
30
15
03 7 15 17
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Noisy Baselines
Possible Causes:
Dirty Flow CellDetector Lamp FailingPulses from Pump if PeriodicTemperature Effects on DetectorAir Bubbles passing through Detector
Time (min.)
28
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Gradient ElutionTemperature Unstable (Refractive Index Detector)Contamination in Mobile PhaseMobile Phase Not in Equilibrium with ColumnContamination Bleed in System
Drifting Baselines
Page 56
Chromatographic Results with “Wrong” Lamp at 214 nm Wavelength
OEM LampOEM Lamp
Lamp from Generic SourceLamp from Generic Source
Tip: Could also be a symptom of aging lamp
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Expanded View of Chromatographic Results Generic Source Lamp at 214 nm Wavelength
OEM Lamp OEM Lamp
Lamp from Generic SourceLamp from Generic Source
Peak 1Peak 1 S/N = 150S/N = 150
Peak 2Peak 2 S/N = 400S/N = 400
Peak 3Peak 3 S/N = 300S/N = 300
Peak 1Peak 1 S/N = 15S/N = 15
Peak 2Peak 2 S/N = 50S/N = 50
Peak 3Peak 3 S/N = 50S/N = 50
Tip: Poor S/N makes it difficult to detect low level impurities
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Effect of Detector Response Time
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time (min)
1.0
Agilent 1100 DADAgilent 1100 WPS with ADVRColumn: Poroshell 300SB-C18
2.1 x 75 mm, 5 mmMobile Phase:A: 95% H2O, 5% ACN with 0.1% TFAB: 5% H2O, 5% ACN with 0.1% TFA
Flow Rate: 2 mL/minTemperature:70°C
Detector: UV 215 nm
Piston stroke: 20 Sample:
1. Neurotensin3. Lysozyme2. RNaseA 4. Myoglobin
0.1 sec
0.2 sec
0.5 sec
1.0 sec
1st peak = 1.2 secAt 20 pts/sec = 24 pts/sec
Response Time
2.0 sec
1st peak = 1.2 secAt 5 pts/sec = 6 pts
• Tip: Adjust the response rate of your detector for best peak detection.
The System is operating well-the settings were poorly made!
Slow Data Rates Can Hinder Impurity Detection and Reduce Sensitivity
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Conclusions
• High pressure (prevention better than the cure) • Undesirable peak shape• Changes in retention/selectivity
Often these problems are not associated with the column and may be caused by instrument and chemistry issues.
•pH of mobile Phase•Instrument Connections•Detector Settings•Metal Contamination
Start With the Correct Questions•Find the Answers•The Answers will Lead to Solutions
HPLC column problems are evident as